Enterococcus faecalis is an important nosocomial pathogen. While a component of the normal human intestinal flora, E. faecalis is a low virulence organism. Yet a variety of infections by E. faecalis occur, including endocarditis, bacteremia, and infections of the urinary track and other tissues. The E. faecalis strains that cause these infections may differ in virulence factors from the docile strains that inhabit the healthy human intestine. Thus an important challenge is to determine the genetic differences between E. faecalis strains with different phenotypes. As a first step toward this goal, this project will determine the genomic sequences of two strains of Enterococcus faecalis, strains OG1RF and HH22, and compare them to the sequence of strain V583, the only other complete E. faecalis genomic sequence. Strain OG1RF is the major experimental strain of E. faecalis, used widely in research for many years, and on which much of the knowledge of E. faecalis biology is based. Strain HH22 is a closer relative of V583, but shows 100-fold greater infectivity in a mouse model. Comparison of these three genomes should provide information as to E. faecalis virulence as well as develop the methodology that can be applied to a wider ranger of strains. Two novel technologies will be used in this study. In the sequencing phase, high genome coverage using the pyrosequencing methodology of 454 Life Sciences will be aimed at reducing the amount of activity needed to bring the genome to a high quality finished grade. Once the sequences are complete, and comparison has been made to V583, the differences determined will be validated using microarrays produced by the oligonucleotide synthesis technology developed at NimbleGen. In addition to these novel methods, standard procedures will be use for shotgun sequencing, finishing, and validation of genomic sequence differences. The introduction of the novel methods will reduce costs and increase throughput, both of which are important for future scale-up to larger numbers of strains.